U.S. patent application number 10/962669 was filed with the patent office on 2006-06-29 for amphiphylic peptide-pna conjugates for the delivery of pna through the blood brain barrier.
Invention is credited to Jehoshua Katzhendler, Itschak Lamensdorf.
Application Number | 20060142227 10/962669 |
Document ID | / |
Family ID | 34435060 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060142227 |
Kind Code |
A1 |
Lamensdorf; Itschak ; et
al. |
June 29, 2006 |
Amphiphylic peptide-PNA conjugates for the delivery of PNA through
the blood brain barrier
Abstract
The invention provides molecules comprising a nucleic acid, a
hydrophobic moiety and a positively charge moiety, useful in the
delivery of a nucleic acid sequence across a cellular membrane. The
invention further relates to the use of these molecules for the
delivery of a nucleic acid sequence to the brain across the blood
brain barrier for diagnostic and therapeutic applications.
Inventors: |
Lamensdorf; Itschak;
(Modiin, IL) ; Katzhendler; Jehoshua; (Jerusalem,
IL) |
Correspondence
Address: |
PEARL COHEN ZEDEK, LLP
1500 BROADWAY 12TH FLOOR
NEW YORK
NY
10036
US
|
Family ID: |
34435060 |
Appl. No.: |
10/962669 |
Filed: |
October 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60510139 |
Oct 14, 2003 |
|
|
|
Current U.S.
Class: |
514/44A ; 514/81;
536/23.2; 544/81 |
Current CPC
Class: |
A61K 47/542 20170801;
A61K 47/554 20170801; C07H 21/04 20130101; A61K 47/543 20170801;
A61K 47/645 20170801; A61K 47/545 20170801; A61K 47/54 20170801;
A61K 48/00 20130101 |
Class at
Publication: |
514/044 ;
514/081; 536/023.2; 544/081 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/04 20060101 C07H021/04; A61K 31/675 20060101
A61K031/675 |
Claims
1. A molecule represented by any one of the formulas I-IV:
[(H).sub.q--(N).sub.r--(P).sub.s].sub.x I.
[(N).sub.r--(H).sub.q--(P).sub.s].sub.x II.
[(P).sub.s--(H).sub.q--(N).sub.r].sub.x III.
[(N).sub.r,(P).sub.s--(H).sub.q].sub.x IV. wherein N is a nucleic
acid sequence in a length of 1-100 bases, H is a hydrophobic moiety
and P is a positively charge moiety; and wherein q is an integer of
1-20, r is an integer of 0-20, s is an integer of 1-25 and x is an
integer of 1-20.
2. The molecule according to claim 1, wherein said nucleic acid
sequence is a mRNA, a cDNA, a DNA, a DNA analog, a polyamide
nucleic acid (PNA), a PNA morpholino, an aminoethylprolyl (aep)
PNA, a pyrrolidinyl PNA, an oligonucleotide, an oligonucleotide
analog, a ribozyme or an RNAi.
3. The molecule according to claim 1, wherein said nucleic acid
sequence is a PNA.
4. The molecule according to claim 2, wherein said nucleic acid
sequence is an antisense, an antigene or a decoy function.
5. The molecule according to claim 1, wherein said nucleic acid
sequence is neutral or negatively charged.
6. The molecule according to claim 1, wherein said hydrophobic
moiety is a nucleic acid.
7. The molecule according to claim 1, wherein said hydrophobic
moiety is hydrophobic peptides, lipid acid, lipid molecules,
octanol, cholesterol, hydrophobic peptide protecting group,
adamantine, pyrene, eicosenoic acid, C.sub.(6-16) glyceride lipid,
phenoxazine, DMT group, cholenic acid, lithocholic acid, myristic
acid, palmitic acid, heptadecyl group, hexadecylglycerol,
geranyloxyhexyl group, hexadecylamine, dihydrotestosterone,
1-pyrene butyric acid, alkanoic acid, alkanol or any derivatives
thereof.
8. The molecule according to claim 7, wherein said alkanoic acid is
represented by the structure R--(CH.sub.2)n--COOH, wherein n=1-20
and R is a linear or branched alkyl.
9. The molecule according to claim 7, wherein said alkanol is
represented by the structure R--(CH.sub.2)n--OH, wherein n=1-20 and
R is a linear or branched alkyl.
10. The molecule according to claim 7, wherein said lipid acid is
undecanoic acid and/or docosahexanenonic acid.
11. The molecule according to claim 7, wherein said hydrophobic
peptide protecting group is Fmoc or Thoc.
12. The molecule according to claim 1, wherein said positively
charge moiety is a nucleic acid.
13. The molecule according to claim 1, wherein said positively
charge moiety is positively charge peptide, peptidomimetic,
polycations, 2-O-aminopropyl, 2-O-dimethylaminopropyl,
2-O-imidazolyl-ethyl, 2-O-aminoethylamino-oxyethyl,
2-dimethylaminoethyl-oxyethyl or any derivative thereof.
14. The molecule according to claim 1, wherein said positively
charge moiety comprises at least one group of arginine, polyamin
and/or guanidine.
15. The molecule according to claim 14, wherein said polyamine is
spermine, spermidine or putricine.
16. The molecule according to claim 1, wherein said hydrophobic
moiety, said nucleic acid sequence and said positively charge
moiety are linked to each other directly via peptide bonds.
17. The molecule according to claim 1, further comprising a linker
moiety linking between said hydrophobic moiety, said nucleic acid
sequence and said positively charge moiety.
18. The molecule according to claim 17, wherein said linker moiety
is polyethylene glycol, disulfide, amide, amine, oxyamine,
oxyimine, morpholine, thioether, thiourea sulfonamide, ether,
ester, carbonate, carbamate, avidin, strepavidin, biotin, praline,
lysine, cysteine, guanidine or any combination thereof.
19. The molecule according to claim 18, wherein the molecular
weight of said polyethylene glycol is in the range of
2000-40,000.
20. A composition comprising as an active ingredient an effective
amount of one or more molecules according to claim 1, together with
one or more pharmaceutically acceptable excipients or
adjuvants.
21. The composition according to claim 20, formulated for oral or
parenteral or intravenous, transdermal, intranasal
administration.
22. A method for delivering a nucleic acid sequence across a
cellular membrane comprising the step of applying to a cell an
effective amount of one or more molecules according to claim 1.
23. The method according to claim 22, wherein said cell is an
endothelial cell, neuronal cell and glial cell.
24. A method for intracellular targeting of a nucleic acid sequence
to an intracellular organelle comprising the step of applying to a
cell an effective amount of one or more molecules according to
claim 1.
25. A method for intracellular targeting of a nucleic acid sequence
to an intracellular organelle comprising the step of applying to a
cell an effective amount of one or more molecules according to
claim 1, wherein said molecule crosses the nuclear membrane.
26. A method for delivering a nucleic acid sequence to the brain
across the blood brain barrier, said method comprising the step of
administering to a subject an effective amount of one or more
molecules according to claim 1.
Description
CROSS REFERENCE DATA
[0001] This Application claims the priority of U.S. Provisional
Application No. 60/510,139 filed Oct. 14, 2003.
FIELD OF THE INVENTION
[0002] The invention provides molecules comprising a nucleic acid,
a hydrophobic moiety and a positively charge moiety, useful in the
delivery of a nucleic acid sequence across a cellular membrane. The
invention further relates to the use of these molecules for the
delivery of a nucleic acid sequence to the brain across the blood
brain barrier for diagnostic and therapeutic applications.
BACKGROUND OF THE INVENTION
[0003] Antisense drugs are small complementary strands of DNA
(oligonucleotides; ODNs) designed to bind to a specific sequence of
nucleotides in the mRNA target, thus inhibiting production of the
encoded protein. Antisense drugs work at early stages in the
disease-causing process, are much more selective, easy to design,
less complex and less expensive than do the traditional drugs.
However, due to their low biomembrane permeability and their
relatively rapid degradation oligonucleotides are generally
considered to be of limited therapeutic value. To improve their
therapeutic applications, the backbone of these antisense compounds
has been chemically modified. The third generation of antisense
chemistry is the polyamide (peptide) nucleic acid (PNA) surrogates.
PNAs are the first successful substitutes of ODNs that have
displayed equal or better binding affinity than natural DNA or RNA
antisense-based drugs. PNAs are hydrophilic macromolecules and
their administration required disruption of plasma membrane.
Therefore, unmodified/naked PNA molecules pass poorly through the
cell membrane and do not have useful therapeutic applications. In
order to improve their cellular uptake PNAs were conjugated to
delivery moieties such as positively charged peptide, receptor
ligands or hydrophobic moiety.
[0004] Several properties of antisense-based drugs suggest that
these compounds will have tremendous potential as future
therapeutics for CNS disorders. As many of the proteins involved in
the pathogenesis of CNS disorders are similar to other (healthy)
vital proteins, and most if not all conventional drugs lack
selectivity for the disease-target proteins a gene specific method
is desirable. In addition, antisense-based drugs inhibit the
production of encoded proteins, by acting at early stages in the
disease-causing process. Since these compounds restrain the
synthesis of the protein, their effect is long lasting and depends
tightly upon the formation of new protein molecules. As most CNS
disorders are chronic, the long-lasting activity of a drug should
provide significant improvement in the patient's compliance, thus
being therapeutically desirable.
[0005] Unfortunately, both ODNs and PNAs cannot cross the
endothelial cellular membrane of the blood brain barrier (BBB)
effectively, thus, preventing the possible use of these
technologies in developing drugs for CNS disorders.
SUMMARY OF THE INVENTION
[0006] In one embodiment, the invention provides a molecule
represented by any one of the formulas I-IV:
[(H).sub.q--(N).sub.r--(P).sub.s].sub.x I.
[(N).sub.r--(H).sub.q--(P).sub.s].sub.x II.
[(P).sub.s--(H).sub.q--(N).sub.r].sub.x III.
[(N).sub.r,(P).sub.s--(H).sub.q].sub.x IV. [0007] wherein N is a
nucleic acid sequence in a length of 1-100 bases, H is a
hydrophobic moiety and P is a positively charge moiety; and [0008]
wherein q is an integer of 1-20, r is an integer of 0-20, s is an
integer of 1-25 and x is an integer of 1-20.
[0009] In one embodiment, the invention further provides a method
for delivering a molecule across a cellular membrane.
[0010] In one embodiment, the invention further provides a method
for delivering a nucleic acid sequence to the brain across the
blood brain barrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The invention will be understood and appreciated more fully
from the following detailed description taken in conjunction with
the appended drawings in which:
[0012] FIG. 1 demonstrates fluorescence images of NMB cells and NMB
cells incubated with fluorescein-labelled peptide-PNA conjugated or
fluorescein-labelled unmodified PNA.
[0013] FIG. 2 demonstrates the uptake of conjugated PNAs into NMB
cells
[0014] FIG. 3 demonstrates the uptake of conjugated PNAs into PC12
cells.
[0015] FIG. 4 demonstrates the uptake of conjugated PNAs into bEND3
cells in different incubation periods (a) and in different
conjugated PNA concentrations (b).
DETAILED DESCRIPTION OF THE INVENTION
[0016] In one embodiment, the invention provides a molecule
represented by any one of the formulas I-IV:
[(H).sub.q--(N).sub.r--(P).sub.s].sub.x V.
[(N).sub.r--(H).sub.q--(P).sub.s].sub.x VI.
[(P).sub.s--(H).sub.q--(N).sub.r].sub.x VII.
[(N).sub.r--(P).sub.s--(H).sub.q].sub.x VIII. [0017] wherein N is a
nucleic acid sequence in a length of 1-100 bases, H is a
hydrophobic moiety and P is a positively charge moiety; and [0018]
wherein q is an integer of 1-20, r is an integer of 0-20, s is an
integer of 1-25 and x is an integer of 1-20.
[0019] In one embodiment of the invention, N is a nucleic acid
sequence in a length of 1-100 bases. In another embodiment, N is a
nucleic acid sequence in a length of 1-10 bases. In another
embodiment, N is a nucleic acid sequence in a length of 1-20 bases.
In another embodiment, N is a nucleic acid sequence in a length of
10-20 bases. In another embodiment, N is a nucleic acid sequence in
a length of 20-30 bases. In another embodiment, N is a nucleic acid
sequence in a length of 30-40 bases. In another embodiment, N is a
nucleic acid sequence in a length of 40-50 bases. In another
embodiment, N is a nucleic acid sequence in a length of 50-100
bases.
[0020] In one embodiment of the invention, q is an integer of 1-20.
In another embodiment, q is an integer of 2-10. In another
embodiment, q is an integer of 6-16. In another embodiment of the
invention, q is 8. In another embodiment of the invention, q is
9.
[0021] In one embodiment of the invention, r is an integer of 0-20.
In another embodiment, r is an integer of 1-10. In another
embodiment, r is an integer of 10-20. In another embodiment, r is
an integer of 2-5.
[0022] In one embodiment of the invention, s is an integer of 1-25.
In another embodiment, s is an integer of 2-15. In another
embodiment, s is an integer of 2-6. In another embodiment, s is
4.
[0023] In one embodiment of the invention, x is an integer of 1-20.
In another embodiment, x is an integer of 2-15. In another
embodiment, s is an integer of 5-10
[0024] In one embodiment of the invention, the nucleic acid
sequence is a mRNA. In another embodiment of the invention, the
nucleic acid sequence is a cDNA. In another embodiment the nucleic
acid sequence is a DNA. In another embodiment of the invention, the
nucleic acid sequence is a DNA analog. In another embodiment of the
invention, the nucleic acid sequence is a PNA. In another
embodiment the nucleic acid sequence is a PNA morpholino. In
another embodiment of the invention, the nucleic acid sequence is
an aminoethylprolyl (aep) PNA. In another embodiment of the
invention, the nucleic acid sequence is a pyrrolidinyl PNA. In
another embodiment of the invention, the nucleic acid sequence is
an oligonucleotide. In another embodiment the nucleic acid sequence
is an oligonucleotide analog. In another embodiment of the
invention, the nucleic acid sequence is a ribozyme. In another
embodiment of the invention, the nucleic acid sequence is an RNAi.
In another embodiment of the invention, the nucleic acid sequence
is a PNA.
[0025] In one embodiment of the invention, nucleic acid sequence is
an antisense. In another embodiment of the invention, the nucleic
acid sequence is an antigene. In another embodiment of the
invention, the nucleic acid sequence is a decoy function. In one
embodiment of the invention, the nucleic acid sequence is neutral.
In another embodiment of the invention, the nucleic acid sequence
is negatively charged. In one embodiment of the invention, nucleic
acid sequence is in antisense orientation to an endogenous
sequence.
[0026] The term "nucleotide" describes a subunit of DNA or RNA
consisting of a nitrogenous base (adenine, guanine, thymine, or
cytosine in DNA; adenine, guanine, uracil, or cytosine in RNA), a
phosphate molecule, and a sugar molecule (deoxyribose in DNA and
ribose in RNA). Thousands of nucleotides are linked to form a DNA
or RNA molecule.
[0027] The term "oligonucleotide" describes a molecule usually
composed of 25 or fewer nucleotides.
[0028] The term "antisense" describes a nucleic acid sequence that
has a sequence exactly opposite to an mRNA molecule made by the
body; binds to the mRNA molecule to prevent a protein from being
made.
[0029] The term "peptide nucleic acids" (PNAs) refers to molecules
that in certain respects are similar to oligonucleotide analogs
however in other very important respects their structure is very
different. In peptide nucleic acids, the deoxyribose phosphate
backbone of oligonucleotides has been replaced with a backbone more
akin to a peptide than a sugar phosphodiester. Each subunit has a
naturally occurring or non-naturally occurring base attached to
this backbone. A non-limiting example is a backbone constructed of
repeating units of N-(2-aminoethyl)glycine or analogues thereof
having a nucleobase attached thereto via a linker such as a
carboxymethyl moiety or analogues thereof to the nitrogen atom of
the glycine portion of the unit. The units are coupled together via
amide bonds formed between the carboxyl group of the glycine moiety
and the amine group of the aminoethyl moiety. The nucleobase can be
one of the four common nucleobases of nucleic acids or they can
include other natural or synthetic nucleobases. Due to the radical
deviation from the deoxyribose backbone, these molecules were named
peptide nucleic acids.
[0030] The term "antigene" refers to molecules, which bind to
double-stranded DNA. Antigenes can enhance or inhibit gene
expression in cells.
[0031] PNA combine peptide chemistry with nucleic acid sequence
biology. In one embodiment, the invention provides a method for
using peptide delivery technologies in the nucleic acid sequence
field. In one embodiment of the invention, the PNA molecule is used
as an antisense moiety, an antigene moiety or a gene modulator
moiety, while in the another embodiment of the invention, the PNA
molecule is used as an apolar peptide-like moiety in an amphiphlic
brain vector.
[0032] In one embodiment, the invention provides a PNA-peptide
conjugate whereas the PNA oligomer is used as an apolar moiety in
the chimeric amphiphilic construct. In one embodiment of the
invention, the molecule is composed from three sequent ional
component: PNA oligomer, hydrophobic and a positively charged
moieties. In one embodiment of the invention, the molecule is
designed to have a positively charged moieties conjugated to the N
or C terminals of hydrophobic modified PNA oligomer.
[0033] In one embodiment of the invention, the hydrophobic moiety
is a nucleic acid. In another embodiment, the hydrophobic moiety is
a nucleic acid analog. In another embodiment of the invention, the
hydrophobic moiety is a hydrophobic peptide. In another embodiment
of the invention the hydrophobic moiety is a lipid acid. In another
embodiment of the invention, the hydrophobic moiety is a lipid
molecules. In another embodiment of the invention, the hydrophobic
moiety is octanol. In another embodiment of the invention, the
hydrophobic moiety is cholesterol. In another embodiment of the
invention, the hydrophobic moiety is a hydrophobic peptide
protecting group. In another embodiment of the invention, the
hydrophobic moiety is adamantine. In another embodiment of the
invention, the hydrophobic moiety is pyrene. In another embodiment
of the invention, the hydrophobic moiety is eicosenoic acid. In
another embodiment of the invention, the hydrophobic moiety is a
C.sub.(6-16) glyceride lipid. In another embodiment of the
invention, the hydrophobic moiety is phenoxazine. In another
embodiment of the invention, the hydrophobic moiety is a DMT group.
In another embodiment of the invention, the hydrophobic moiety is
cholenic acid. In another embodiment of the invention, the
hydrophobic moiety is lithocholic acid. In another embodiment of
the invention, the hydrophobic moiety is myristic acid. In another
embodiment of the invention, the hydrophobic moiety is palmitic
acid. In another embodiment of the invention, the hydrophobic
moiety is a heptadecyl group. In another embodiment of the
invention, the hydrophobic moiety is hexadecylglycerol. In another
embodiment of the invention, the hydrophobic moiety is a
geranyloxyhexyl group. In another embodiment of the invention, the
hydrophobic moiety is hexadecylamine. In another embodiment of the
invention, the hydrophobic moiety is dihydrotestosterone. In
another embodiment of the invention, the hydrophobic moiety is
1-pyrene butyric acid. In another embodiment of the invention, the
hydrophobic moiety is alkanoic acid. In another embodiment of the
invention, the hydrophobic moiety is alkanol. In another embodiment
of the invention, the hydrophobic moiety is and any derivatives of
the above mentioned moieties. In one embodiment of the invention,
the alkanoic acid is represented by the structure
R--(CH.sub.2)n--COOH, wherein n is an integer of 1-20 and R is a
linear or branched alkyl. In another embodiment of the invention, n
is an integer of 6-16. In one embodiment of the invention, the
alkanol is represented by the structure R--(CH.sub.2)n--OH, wherein
n an integer of 1-20 and R is a linear or branched alkyl. In
another embodiment of the invention, n is an integer of 6-16. In
one embodiment of the invention, the lipid acid is undecanoic acid.
In another embodiment of the invention, the lipid acid is
docosahexanenonic acid. In one embodiment of the invention, the
hydrophobic peptide protecting group is Fmoc. In another embodiment
of the invention, the hydrophobic peptide protecting group is
Tboc.
[0034] As contemplated herein, an "alkyl" group refers to a
saturated aliphatic hydrocarbon, including straight-chain,
branched-chain and cyclic alkyl groups. In an embodiment of the
invention, the alkyl group has 1-4 carbons. In another embodiment,
the alkyl group is a methyl group. In another embodiment, the alkyl
group is an ethyl group. In another embodiment, the alkyl group is
a propyl group. In another embodiment, the alkyl group is a butyl
group. The alkyl group may be unsubstituted or substituted by one
or more groups selected from halogen, hydroxy, alkoxy carbonyl,
amido, alkylamido, dialkylamido, nitro, amino, alkylamino,
dialkylamino, carboxyl, thio and thioalkyl.
[0035] In one embodiment of the invention, the positively charge
moiety is a nucleic acid sequence or a nucleic acid analog. In
another embodiment, the positively charge moiety is a positively
charge peptide. In another embodiment, the positively charge moiety
is a peptidomimetic. In another embodiment, the positively charge
moiety is a polycations. In another embodiment, the positively
charge moiety is 2-O-aminopropyl. In another embodiment, the
positively charge moiety is 2-O-dimethylaminopropyl. In another
embodiment, the positively charge moiety is 2-O-imidazolyl-ethyl.
In another embodiment, the positively charge moiety is
2-O-aminoethylamino-oxyethyl. In another embodiment, the positively
charge moiety is 2-dimethylaminoethyl-oxyethyl. In another
embodiment, the positively charge moiety is and any derivative of
the above mentioned moieties. In another embodiment, the positively
charge moiety is arginine. In another embodiment, the positively
charge moiety is D-arginine. In another embodiment, the positively
charge moiety is polyarginine. In another embodiment, the
positively charge moiety is polyamine. In another embodiment, the
positively charge moiety is guanidine. In another embodiment, the
polyamine is spermine. In another embodiment, the polyamine is
spermidine. In another embodiment, the polyamine is putricine.
[0036] In one embodiment of the invention, the hydrophobic moiety,
the nucleic acid sequence and the positively charge moiety are
linked to each other directly via peptide bonds.
[0037] In one embodiment, the invention provides a molecule
comprising undecanoic acid, a PNA and at least one arginine group.
In another embodiment, the undecanoic acid is directly or
indirectly linked, on one side to the PNA and on the other side to
at least one arginine group. In another embodiment, the PNA is
directly or indirectly linked, on one side to the undecanoic acid
and on the other side to at least one arginine group.
[0038] In one embodiment, the invention provides a molecule
comprising undecanoic acid, a PNA, morpholino or any neutral
nucleic acid analogs and at least one arginine group. In another
embodiment, the undecanoic acid is directly or indirectly linked,
on one side to the PNA, morpholino or any neutral nucleic acid
analogs and on the other side to at least one arginine group. In
another embodiment, the PNA morpholino is directly or indirectly
linked, on one side to the undecanoic acid and on the other side to
at least one arginine group.
[0039] In one embodiment, the invention provides a molecule
comprising undecanoic acid, an oligonucleotide and at least one
arginine group. In another embodiment, the undecanoic acid is
directly or indirectly linked, on one side to the oligonucleotide
and on the other side to at least one arginine group. In another
embodiment, the oligonucleotide is directly or indirectly linked,
on one side to the undecanoic acid and on the other side to at
least one arginine group. In one embodiment of the invention, the
oligonucleotide is an amphiphilic oligonucleotide.
[0040] In one embodiment, the invention provides a molecule
comprising a PNA, an antisense moiety and at least one arginine
group. In another embodiment, the PNA is directly or indirectly
linked, on one side to the antisense moiety and on the other side
to at least one arginine group. In another embodiment, the
antisense moiety is directly or indirectly linked, on one side to
the PNA and on the other side to at least one arginine group.
[0041] In one embodiment, the invention provides a molecule
comprising a PNA, an antisense moiety and at least one arginine
group. In another embodiment, the PNA is directly or indirectly
linked, on one side to the antisense moiety and on the other side
to at least one arginine group. In another embodiment, the
antisense moiety is directly or indirectly linked, on one side to
the PNA and on the other side to at least one arginine group.
[0042] In one embodiment, the invention provides a molecule
comprising an oligonucleotide, an antisense moiety and at least one
arginine group. In another embodiment, the oligonucleotide is
directly or indirectly linked, on one side to the antisense moiety
and on the other side to at least one arginine group. In another
embodiment, the antisense moiety is directly or indirectly linked,
on one side to the oligonucleotide and on the other side to at
least one arginine group. In one embodiment of the invention, the
oligonucleotide is an amphiphilic oligonucleotide.
[0043] In one embodiment, the invention provides a molecule
comprising undecanoic acid, a PNA and at least one polyamine group.
In another embodiment, the undecanoic acid is directly or
indirectly linked, on one side to the PNA and on the other side to
at least one polyamine group. In another embodiment, the PNA is
directly or indirectly linked, on one side to the undecanoic acid
and on the other side to at least one polyamine group. In another
embodiment, the polyamine is spermidine. In another embodiment, the
polyamine is putricine.
[0044] In one embodiment, the invention provides a molecule
comprising undecanoic acid, a PNA and at least one guanidinium
group. In another embodiment, the undecanoic acid is directly or
indirectly linked, on one side to the PNA and on the other side to
at least one guanidinium group. In another embodiment, the PNA is
directly or indirectly linked, on one side to the undecanoic acid
and on the other side to at least one guanidinium group.
[0045] In one embodiment, the invention provides a molecule
comprising docosahexanenonic acid (DHA)-PNA (1-20
bases)-(Arg).sub.4. In one embodiment, the invention provides a
molecule comprising undecanoic acid --PNA (1-20 bases)-(Arg).sub.4.
In one embodiment, the invention provides a molecule comprising
PNA-DHA-(Arg).sub.4. In one embodiment, the invention provides a
molecule comprising undecanoic acid-CCGCTCCG (SEQ ID NO.
1)-(Arg).sub.2-15. In one embodiment, the invention provides a
molecule comprising docosahexanenonic acid-CAT GGT GGA CGT (SEQ ID
NO. 2)-(Arg).sub.2-15. In one embodiment, the invention provides a
molecule comprising undecanoic acid-CAT GGT GGA CGT (SEQ ID NO.
3)-(Arg).sub.2-15. In one embodiment, the invention provides a
molecule comprising docosahexanenonic acid-CTT TCT CCT TTT CC (SEQ
ID NO. 4)-(Arg).sub.2-15. In one embodiment, the invention provides
a molecule comprising undecanoic acid --CTT TCT CCT TTT CC (SEQ ID
NO. 4)-(Arg).sub.2-15. In one embodiment of the invention, the PNA
is in a length of 5-10 bases.
[0046] In one embodiment, the invention provides a molecule
comprising DHA (SEQ ID NO. 5)-PNA (0-20 bases)-(deoxynucleic
guanidine-DNG).sub.1-20. In one embodiment, the invention provides
a molecule comprising PNA (0-20 bases)-DHA-(DNG).sub.1-20 In one
embodiment, the invention provides a molecule comprising DHA-PNA
(0-20 bases)-(aminoethylprolyl (aep) PNA).sub.1-20 In one
embodiment, the invention provides a molecule comprising PNA (0-20
bases)-DHA-(aepPNA).sub.1-20 In one embodiment, the invention
provides a molecule comprising (Pyrrolidinyl PNA).sub.1-20-PNA
(0-20 bases)-(aepPNA).sub.1-20 In one embodiment, the invention
provides a molecule comprising PNA (0-20 bases)-(Pyrrolidinyl
PNA).sub.1-20-(aepPNA).sub.1-20. In one embodiment of the
invention, the PNA is in a length of 5-10 bases.
[0047] In one embodiment, the invention provides a molecule
comprising TACTCATGGGCACACT (SEQ ID NO. 6)-FLFLRR (SEQ ID NO. 7).
In one embodiment, the invention provides a molecule comprising
(C).sub.8-TTT GCT CTT ACT CAT (SEQ ID NO. 8)-(D-Arg).sub.6. In one
embodiment, the invention provides a molecule comprising
PEG-(C).sub.8-TTT GCT CTT ACT CAT (SEQ ID NO. 8)-(D-Arg).sub.6. In
one embodiment, the invention provides a molecule comprising
PEG-TTT GCT CTT ACT CAT (SEQ ID NO. 8)-(C).sub.8-- (D-Arg).sub.6.
In one embodiment, the invention provides a molecule comprising TTT
GCT CTT ACT CAT (SEQ ID No. 8)-PEG-(C).sub.8-- (D-Arg).sub.6. In
one embodiment, the invention provides a molecule comprising
(Arg).sub.2-TACTCATGGGCACACT (SEQ ID NO. 9)-FLFLFL (SEQ ID NO. 10).
In one embodiment, the invention provides a molecule comprising
(Arg).sub.6-TACTCATGGGCACACT (SEQ ID NO. 9)-FLFLFL (SEQ ID NO. 10).
In one embodiment, the invention provides a molecule comprising TTT
GCT CTT ACT CAT (SEQ ID NO. 8)-(FL).sub.2-3 (SEQ ID NO.
11)-(Arg).sub.2-6. In one embodiment, the invention provides a
molecule comprising (FL).sub.2-3 (SEQ ID NO. 11)-TTT GCT CTT ACT
CAT (SEQ ID NO. 8)-(Arg).sub.2-6. In one embodiment, the invention
provides a molecule comprising CHKKKKKKHC (SEQ ID NO. 12)-PNA (1-20
bases)-(FL).sub.2-3 (SEQ ID NO. 11). In one embodiment, the
invention provides a molecule comprising CHKKKKKKHCC (SEQ ID NO.
13) PNA (1-20 bases)-(FL).sub.2-3 (SEQ ID NO. 11). In one
embodiment, the invention provides a molecule comprising
(CH.sub.2).sub.9--CO-(CCGCTCCG) (SEQ ID NO. 15)-GRRRK (SEQ ID NO.
16). We note that (CH.sub.2).sub.9--CO is a chemical structure. In
one embodiment of the invention, the PNA is in a length of 5-10
bases.
[0048] In one embodiment, the invention provides a molecule
comprising DHA-PNA (1-20 bases)-putricine. In one embodiment, the
invention provides a molecule comprising PNA (1-20
bases)-DHA-putricine. In one embodiment, the invention provides a
molecule comprising undecanoic acid-PNA (1-20 bases)-Putricine. In
one embodiment, the invention provides a molecule comprising PNA
(1-20 bases)-undecanoic acid-putricine. In one embodiment, the
invention provides a molecule comprising undecanoic-PNA (1-20
bases)-spermine. In one embodiment, the invention provides a
molecule comprising PNA (1-20 bases)-undecanoic acid-spermine. In
one embodiment, the invention provides a molecule comprising
undecanoic acid -PNA (1-20 bases)-spermidine. In one embodiment,
the invention provides a molecule comprising PNA (1-20
bases)-undecanoic acid-spermidine. In one embodiment of the
invention, the PNA is in a length of 5-10 bases.
[0049] In one embodiment, the invention provides a molecule
comprising Fmoc-PNA(1-20 bases)-putricine. In one embodiment, the
invention provides a molecule comprising. TBoc-PNA (1-20
bases)-putricine. In one embodiment, the invention provides a
molecule comprising docosahexanenonic acid-PNA (1-20
bases)-Diethylenetriamine. In one embodiment, the invention
provides a molecule comprising docosahexanenonic acid-PNA (1-20
bases)-polyethylenimine. In one embodiment, the invention provides
a molecule comprising allyl substituted PNA (1-20
bases)-polyethylenimine. In one embodiment, the invention provides
a molecule comprising chloro and/or bromo-halogenated PNA (1-20
bases)-polyethylenimine. In one embodiment, the invention provides
a molecule comprising allyl substituted PNA (1-20
bases)-(Arg/D-Arg).sub.2-10. By Arg/D-Arg) it is meant, either
arginine or the D-isomer of arginine (D-Arg), in any combination
thereof. In one embodiment, the invention provides a molecule
comprising chloro and/or bromo-halogenated substituted PNA (1-20
bases)-(Arg/D-Arg).sub.2-10. In one embodiment of the invention,
the PNA is in a length of 5-10 bases. In one embodiment of the
invention, the number of Arg/D-Arg groups is 2-6. In another
embodiment, the number of Arg/D-Arg groups is 4.
[0050] In one embodiment of the invention, the molecule further
comprising a linker moiety linking between the hydrophobic moiety,
the nucleic acid sequence and the positively charge moiety. In
another embodiment, the linker moiety is polyethylene glycol (PEG).
In another embodiment, the molecular weight of said PEG is in the
range of 2000-40,000. In another embodiment, the linker moiety is a
disulfide. In another embodiment, the linker moiety is an amide. In
another embodiment, the linker moiety is an amine. In another
embodiment, the linker moiety is an oxyamine. In another
embodiment, the linker moiety is an oxyimine. In another
embodiment, the linker moiety is a morpholine. In another
embodiment, the linker moiety is a thioether. In another
embodiment, the linker moiety is thiourea sulfonamide. In another
embodiment, the linker moiety is an ether. In another embodiment,
the linker moiety is an ester. In another embodiment, the linker
moiety is a carbonate. In another embodiment, the linker moiety is
a carbamate. In another embodiment, the linker moiety is guanidine.
In another embodiment, the linker moiety is avidin. In another
embodiment, the linker moiety is strepavidin. In another
embodiment, the linker moiety is biotin. In another embodiment, the
linker moiety is praline. In another embodiment, the linker moiety
is lysine. In another embodiment, the linker moiety is
cysteine.
[0051] In one embodiment of the invention, the liable linker or
peptide bond to polyethylene glycol conjugated to the molecule
improves phramacokinetic properties and overcomes possible side
effects induced by the amphiphilic PNA. In one embodiment of the
invention, the linker is conjugated to the molecule via a known
technology.
[0052] In one embodiment, the invention provides a molecule
comprising CHKKKKKKHCC (SEQ ID. No. 17)-PNA (1-20
bases)-(FL).sub.2-3 (SEQ ID NO. 11). In one embodiment, the
invention provides a molecule comprising CHKKKKKKHCC (SEQ ID. No.
17)-PNA (1-20 bases)-avidin/strepavidin-biotin-(FL).sub.2-3 (SEQ ID
NO. 11). In one embodiment, the invention provides a molecule
comprising CHKKKKKKHCC (SEQ ID. No. 17) PNA (1-20
bases)-(FL).sub.2-3 (SEQ ID NO. 11). In one embodiment, the
invention provides a molecule comprising CHKKKKKKHCC (SEQ ID. No.
17) PNA (1-20 bases)-(FL).sub.2-3 (SEQ ID NO. 11). In one
embodiment, the invention provides a molecule comprising
(Arg/D-Arg).sub.2-6-PK (SEQ ID NO. 20)-CGC TGG GC (SEQ ID NO. 19)
CC(FL).sub.2-3 (SEQ ID NO. 18). In one embodiment, the invention
provides a molecule comprising (Arg/D-Arg).sub.2 6-CGC TGG GC (SEQ
ID NO. 20) CC(FL).sub.2-3 (SEQ ID NO. 18). In one embodiment, the
invention provides a molecule comprising (Arg).sub.2-6-PEG-CGC TGG
GC (SEQ ID NO. 20) CC(FL).sub.2-3 (SEQ ID NO. 18). In one
embodiment, the invention provides a molecule comprising
(Arg/D-Arg).sub.2-6-PEG-C--C (SEQ ID NO. 21)-CGC TGG GC (SEQ ID NO.
20)-CC(FL).sub.2-3 (SEQ ID NO. 18). In one embodiment of the
invention, the PNA is in a length of 5-10 bases. In one embodiment
of the invention, the number of Arg/D-Arg groups is 2-6. In another
embodiment, the number of Arg/D-Arg groups is 4. In one embodiment,
the invention the molecular weight of said polyethylene glycol is
in the range of 2000-40,000.
[0053] In one embodiment, the invention further provides a
composition comprising as an active ingredient an effective amount
of one or more molecules according to claim 1, together with one or
more pharmaceutically acceptable excipients or adjuvants. In one
embodiment of the invention, the composition is formulated for oral
or parenteral administration. In another embodiment, the
composition is formulated as uncoated tablets, coated tablets,
pills, capsules, powder or suspension. In another embodiment, the
composition is formulated for intravenous administration. In
another embodiment, the composition is formulated intranasal
administration. In another embodiment, the composition is
formulated administration via aerosols. In another embodiment, the
composition is formulated for transdermal administration. In
another embodiment, the composition is formulated in an ointment,
cream or gel form. In another embodiment, the compositions of the
invention are formulated in a liquid dosage form. Examples of
suitable liquid dosage forms include solutions or suspensions in
water, pharmaceutically acceptable fats and oils, alcohols or other
organic solvents, including esters, emulsions, syrups or elixirs,
solutions and/or suspensions.
[0054] Suitable excipients and carriers can be solid or liquid and
the type is generally chosen based on the type of administration
being used. Liposomes may also be used to deliver the composition.
Examples of suitable solid carriers include lactose, sucrose,
gelatin and agar. Oral dosage forms may contain suitable binders,
lubricants, diluents, disintegrating agents, coloring agents,
flavoring agents, flow-inducing agents, and melting agents. Liquid
dosage forms may contain, for example, suitable solvents,
preservatives, emulsifying agents, suspending agents, diluents,
sweeteners, thickeners, and melting agents parenteral and
intravenous forms should also include minerals and other materials
to make them compatible with the type of injection or delivery
system chosen.
[0055] In one embodiment, the invention provides a method for the
synthesis of a molecule according to the invention.
[0056] In one embodiment, the invention further provides a method
for delivering a nucleic acid sequence across a cellular membrane
comprising the step of applying to a cell an effective amount of
one or more molecules according to the invention. In one embodiment
of the invention, the cell is an endothelial cell. In another
embodiment, the cell is a neuronal cell. In another embodiment, the
cell is a glial cell. In another embodiment, the cell is a muscle
cell.
[0057] In one embodiment, the invention provides a method for the
improved delivery of PNAs into mammalian cells. In one embodiment,
the invention provides an amphiphilic PNA chimeric moiety with
improved neuronal, endothelial delivery properties.
[0058] In one embodiment, the invention further provides a method
for intracellular targeting of a nucleic acid sequence to an
intracellular organelle comprising the step of applying to a cell
an effective amount of one or more molecules according to the
invention. In one embodiment of the invention, charge distribution,
length of the apolar nucleic acid chain and hydrophobicity can
affect sub-cellular compartization. In one embodiment, the
invention provides a method for intracellular targeting of a
nucleic acid sequence to an intracellular organelle comprising the
step of applying to a cell an effective amount of one or more
molecules of the invention, wherein the molecules crosse the
nuclear membrane.
[0059] In one embodiment, the invention further provides a method
for delivering a nucleic acid sequence to the brain across the
blood brain barrier, said method comprising the step of
administering to a subject an effective amount of one or more
molecules according to the invention. In another embodiment, the
invention provides a method for delivering a nucleic acid sequence
to the brain across the blood brain barrier, said method comprising
the step of administering to a subject a composition according to
the invention.
[0060] In one embodiment of the invention, polyarginine oligomers
are used to improve BBB penetration of PNA-based constructs In
another embodiment, arginine guanido groups are used to improve BBB
penetration of PNA-based constructs. In one embodiment of the
invention, the increased brain uptake through the BBB is via
guanidine basic amino acid transporters. In another embodiment, the
increased brain uptake is the result of augmented AMT as a result
of increased permeability surface due to the presence of positive
charge.
[0061] In one embodiment of the invention, polyamines are used to
improve BBB penetration of PNA-based constructs. In another
embodiment, polyamine is putrescine. In another embodiment,
polyamine is sperimidine. In another embodiment, polyamine is
putrescine. In another embodiment, polyamine is spermine. In one
embodiment of the invention, the increased brain uptakethrough the
BBB is via polyamine transporters. In another embodiment, the
increased brain uptake is the result of augmented AMT as a result
of increased permeability surface due to the presence of positive
charge.
[0062] In one embodiment, the invention further provides a method
for delivering a gene across the blood brain barrier for expression
in the brain, said method said method comprising administering to a
subject an effective amount of one or more molecules according to
the invention. In another embodiment, the invention provides a
method for delivering a gene across the blood brain barrier for
expression in the brain, said method said method comprising
administering to a subject one a composition according to the
invention.
[0063] In one embodiment, the invention further provides a method
for modulating gene expression, said method said method comprising
administering to a subject an effective amount of one or more
molecules according to the invention. In another embodiment, the
invention provides a method for modulating gene expression, said
method said method comprising administering to a subject one a
composition according to the invention.
[0064] In one embodiment, the invention provides a kit comprising
an effective amount of one or more molecules according to the
invention. In another embodiment, the kit allows gene labeling. In
another embodiment, the kit further comprising labeling and/or
reaction buffers. In another embodiment, the molecule is conjugated
to a fluorescent label, a calorimetric label, a radiolabel label or
a chemical label.
[0065] In one embodiment, the invention further provides a method
for the treatment, prevention and control of a disease, said method
comprising administering to a subject an effective amount of one or
more molecules according to the invention. In another embodiment,
the invention provides a method for the treatment, prevention and
control of a disease, said method comprising administering to a
subject a composition according to the invention. In one embodiment
of the invention, the disease is a central nervous system related
disease.
[0066] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention,
suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control.
[0067] This invention is further illustrated in the Experimental
Details section, which follows. This section is set forth to aid in
an understanding of the invention but is not intended to, and
should not be construed to, limit in any way the invention as set
forth in the claims that follow thereafter.
EXPERIMENTAL DETAILS SECTION
Methods
Synthesis:
PNA and Peptide Synthesis.
[0068] Oligomers were made on a Chemspeed Automatic Synthesizer on
5 micromole scale applying double coupling of
fluorenylmethoxycarbonyl (Fmoc) protected nucleobases monomers or
amino-acids (AA) with a ratio of 1:3 each coupling cycle. Resin
used was either wang resin (0.57 mmole/g) or TGA resin 9 Novasyn,
0.15 mmole/g) already loaded with amino acid. Total reaction volume
was 250 microliter and coupling reagent utilized was either BOP or
HBTU in the presence of diisopropylethylamine and 2,6-lutidine.
PNAs or PNAs combined with peptides or/and with 11-amino undecanoic
acid were deprotected and removed from the resin by trifluoro
acetic acid 1 ml containing triethylsilyl (2 h). Product was
percipitate with cold diethylether 14 ml collected and subjected to
RP-HPLC on a 10 micron vydac column at 50.degree. C. Gradient 10%
to 90% acetonitril (CAN) in 25 min, rt.about.10 min. Analysis:
Mass-spectra: MALDI-TOF
Cell Culture:
[0069] As an in vitro models for neurons and for BBB we used the
human neuroblastoma NMB cell line the rat catecholaminergic cell
line PC 12 and the mice brain-derived endothelial cell line
bEND3.
[0070] NMB cells were adapted to grow in Dulbecco modified Eagle
medium (DMEM) with 10% Hyclone calf serum. PC12 cells were grown in
DMEM, 10% horse serum and 5% calf serum. bEND3 cells were grown in
DMEM, %10 calf serum supplemented with 2 mM glutamine. Cells were
maintained in a 10% CO.sub.2 humidified incubator at 37.degree. C.
Cells were routinely sub-cultured every four to five days. When
cells (5.times.10.sup.3) were plated in 96 wells, dishes were
pretreated with poly-L-ornithine.
Uptake Experiments:
[0071] Uptake of PNA and conjugated PNA was determined as
previously published [4,9] by measurement of fluorescence retained
in cells following incubation with fluorescein-labelled peptide-PNA
conjugated or fluorescein-labelled unmodified PNA. The molecules
were incubated with NMB. After incubation, the cells were
extensively washed with ice-cold PBS followed by acid wash (1.0 M
NaCl/0.4 M NaOAc, PH 3.3). PC12 or bEND3 for different time period.
A procedure that was found to removed both noninternalized oligomer
and dead cells. After the final wash intra-cellular fluorescence
was determined by fluoremeter (FLUOstar BMG Labtechnologies).
EXAMPLES
Example 1
Fluorescence Microscopy
[0072] To test neuronal compartization and cellular distribution of
unmodified PNA. The human neuronal cell line NMB cells were seeded
on a poly-L-ornithine coated 35 mm dish. 24 hours following seeding
the cell culture medium was replaced with DMEM containing 1 .mu.M
PNA. Cells were incubated with FITC labeled PNA and propidium
iodide (PI) for nucleolus staining for 4 hours. Following
incubation cells were washed 3 times with PBS and medium was
replaced with fresh DMEM. Bright field and UV images were taken
using Olympus BH-2 microscope. Images were digitized using the
Power PC i.view 32 image analysis software (FIG. 1). As can be seen
neuronal cell line are taking up unmodified PNA in a punctuated
manner indicating that unmodified PNA are being tramped in the
endosomal, lysosomal system.
Example 2
NMB Cells
[0073] To test the effect of amphilic structure composition uptake
of conjugated PNAs into the human neuronal cell line NMB was
determined. Cells were seeded on 96 well dishes coated with
poly-1-ornithine. Day after seeding medium was replaced with fresh
medium without serum containing one micromolar PNAs. Following
one-hour incubation medium was removed and cells were washed three
times with acid wash solution and fluorescent determined (FIG. 2).
P represents PNA, U represents undecanoic acid; R represents
arginine; and (n) the number of arginine groups. The control group
represents cells treated with PNA. Adding hydrophobic moiety
increases PNA uptake. However, addition of amphiphylic moiety
further increase PNA uptake. The highest intracellular content
could be achieved when the PNA sequence was used as a neutral
spacer between the hydrophobic and the positively charge
components.
Example 3
PC12 Cells
[0074] To test dose response and to optimize amphiphylic
composition several modified PNAs were tested on PC12 cell line.
Uptake of conjugated PNAs into the neuronal cell line PC12. Cells
were seeded on 96 well dishes coated with poly-1-lysine. Day after
seeding medium was replaced with fresh medium without serum
containing different concentrations (1-4 .mu.M) of PNAs. Following
one-hour incubation medium was removed and cells were washed three
times with acid wash solution and fluorescent determined (FIG. 3).
P, U and R(n) are defined above. PNA uptake increase in a dose
dependent manner. Amphiphilic modifications markedly enhanced PNA
uptake. Changing the number of arginine residues affected modified
PNA uptake as increasing the number of arginine from 4 to 6
monomers decreases neuronal uptake.
Example 4
bEND3 Cells
[0075] The BEND3 cell line (a murine cell line model of BBB) was
used to test time and dose response dependency of modified PNA
(Amphiphilic based modifications). Uptake of conjugated PNAs into
the mice endothelial brain cell line bEND3. Cells were seeded on 96
well dishes coated with poly-1-ornithine. Day after seeding medium
was replaced with fresh medium without serum containing one
micromolar PNAs. Cells were incubated for different time period of
15 to 60 minutes. Following incubation medium was removed and cells
were washed three times with acid wash solution with acid wash
solution and fluorescent determined (FIG. 4a), P, U and R(n) are
defined above. As can be seen UPR(4) had a higher uptake than the
control or the PU indicating that amphipilic modification can
increase penetration through the BBB.
[0076] Cells were also incubated with different concentrations of
conjugated PNAs. Following incubation medium was removed and cells
were washed three times with acid wash solution with acid wash
solution and fluorescent determined (FIG. 4b). P, U and R(n) are
defined above. In an excellent agreement with the neuronal cell
line finding, also in the BBB model UPR(4) composition was found to
be the most potent conjugation.
[0077] It will be appreciated that the invention is not limited by
what has been described hereinabove and that numerous
modifications, all of which fall within the scope of the invention,
exist. Rather the scope of the invention is defined by the claims
that follow:
Sequence CWU 1
1
15 1 8 DNA Homo sapiens 1 ccgctccg 8 2 12 DNA Homo sapiens 2
catggtggac gt 12 3 14 DNA Homo sapiens 3 ctttctcctt ttcc 14 4 16
DNA Homo sapiens 4 tactcatggg cacact 16 5 6 PRT Homo sapiens 5 Phe
Leu Phe Leu Arg Arg 1 5 6 15 DNA Homo sapiens 6 tttgctctta ctcat 15
7 16 DNA Homo sapiens 7 tactcatggg cacact 16 8 6 PRT Homo sapiens 8
Phe Leu Phe Leu Phe Leu 1 5 9 10 PRT Homo sapiens 9 Cys His Lys Lys
Lys Lys Lys Lys His Cys 1 5 10 10 8 DNA Homo sapiens 10 ccgctccg 8
11 5 PRT Homo sapiens 11 Gly Arg Arg Arg Lys 1 5 12 2 PRT Homo
sapiens 12 Pro Lys 1 13 8 DNA Homo sapiens 13 cgctgggc 8 14 6 PRT
Homo sapiens 14 Cys Cys Phe Leu Phe Leu 1 5 15 8 PRT Homo sapiens
15 Cys Cys Phe Leu Phe Leu Phe Leu 1 5
* * * * *